DNA-binding polyamide drug conjugates

ABSTRACT

A conjugate of formula: V—(Y) a —Z-T: (I), T-X—B—(Y) a —Z-T′: (II), V—(Y) a  Z—(Y′) a  V′: (III), T-X—B—(Y) a —Z—(Y′) a —X′—B′-T′: (IV), V—(Y) a —Z—(Y′) a —X—B-T: (V), V—(Y) a —Z—X—B—Z′—(Y′) a —(V′) b : (VI), or (W) a —(Y) b —[(Z) c —(Y′) d —(X—B) e —(Y″) f —(Z′) g ] h —(Y . . . ) i —(W′) j : (VII), in which W and W′ are independently a DNA intercalator or terminal subunit, V and V′ are independently a DNA intercalator, X and X′ are independently a DNA alkylator, B and B′ are the same or different and each is a heteoaromatic residue that is attached to the Nterminus of an alkylator subunit (X or X′), Y, Y′, Y″ and Y′″ are independently a linker, T and T′ are independently terminal subunits, Z and Z′ are independently a polyamide group that binds to the minor groove of DNA, a, b, c, d, f, g, i, and j are independently 0 to 5, and e and h are independently 1 to 5, a composition comprising a conjugate of any of formulae (I)-(VII) and a carrier, and a method for treating cancer in a mammal comprising administering an effective amount of a conjugate of any of formulae (I)-(VII) or a composition comprising same.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is the U.S. national phase of International PatentApplication No. PCT/US03/06006, which was filed on Feb. 27, 2003, andwhich claims the benefit of both U.S. Provisional Patent Application No.60/361,050, which was filed on Feb. 27, 2002, and U.S. ProvisionalPatent Application No. 60/370,168, which was filed on Apr. 5, 2002.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to conjugates comprising one or more DNAintercalator(s) and/or one or more DNA alkylator(s) linked to one ormore polyamide DNA minor groove binder(s). The conjugates of the presentinvention and compositions thereof are useful in the treatment of cancerin a mammal.

BACKGROUND OF THE INVENTION

Specificity is one of the goals of modern drug design. It is known thatDNA is a target of most antitumor drugs, since many antitumor drugs areeffective by inhibiting nucleic acid (DNA or RNA) or protein synthesis.There are many potent antitumor compounds with a wide spectrum ofactivities against many tumor cell lines, both in vitro and in vivo, butbecause these compounds are so toxic, normal cells and tissue can beadversely affected as well. Many researchers have proposed ideas ofincreasing selectivity, while maintaining a high degree of toxicity.

Polyamides that contain polypyrrole carboxamide and/or polyimidazolecarboxamide subunits are known to bind specifically to the minor grooveof DNA (see, for example, Dervan, Bioorganic and Medicinal Chemistry, 9:2215-2235 (2001); Soto et al., Nucleic Acids Research, 29(17): 3638-3645(2001); and Reddy et al., Current Medicinal Chemistry, 8: 475-508(2001)). Such polyamides can be designed so that they bind to DNA in asequence-specific manner. These polyamide minor groove binders caninhibit or suppress gene functions. The polyamide minor groove bindersbis-lexitropsins (Reddy et al., Current Medicinal Chemistry, 8: 475-508(2001)), for example, have shown enhanced cytotoxic activity against KBhuman nasopharyngeal carcinoma. It also has been shown thatdouble-stranded hairpin polyamides can permeate cellular and nuclearmembranes of eukaryotes and, when targeted to promoter regions, caninhibit specific gene expression (Gottesfeld et al., Nature, 387:202-205 (1997); and Dickinson et al., Proc. Natl. Acad. Sci. U.S.A., 95:12890-12895 (1998)). In addition to the double-stranded hairpinpolyamides, the single-stranded analogues have been proven to carry outinhibition of gene expression in Drosophila (Maeshima et al., The EMBOJournal, 20: 3218-3228 (2001)).

Researchers have linked toxic compounds to polyamide sequences, such asnetropsin, distanycin and lexitropsin (see, for example, Chang et al.,J. Am. Chem. Soc., 122: 4856-4864 (2000); Gupta et al., Anti-Cancer DrugDesign, 11(8): 581-596 (1996); Jia et al., Heterocyclic Commun., 4(6):557-560 (1998); Jia et al., Chem. Commun, (2): 119-120 (1999); Jia etal., Synlett, (5): 603-606 (2000); and Wang et al., Gene, 149(I): 63-67(1994)). While these conjugates may show some antitumor activity, theconjugates, themselves, have the wrong geometric and/or electronicparameters that hinder the fit in the minor groove. A poor fit canresult in a lower efficacy or selectivity as well as higher side effectsdue to nonspecific binding to untargeted genomic elements or DNAsequences.

Thus, there still exists a need for therapeutic conjugates that haveimproved antitumor selectivity and DNA sequence-specific bindingproperties. Ideally, these conjugates would elicit fewer side effectsand less damage to healthy cells and tissue. Effective therapeuticconjugates can be designed rationally, because an understanding of thegeometry of the conjugates enables a better fit of the drug into theshape of the minor groove pocket, thereby increasing thesequence-specificity. The present invention provides such therapeuticconjugates. The conjugates of the present invention bind in the minorgroove of DNA in a sequence-specific manner and effectively deliver atoxic moiety.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a conjugate of formula:V—(Y)_(a)-Z-T  (I) orT-X—B—(Y)_(a)-Z-T′  (II),in which V is a DNA intercalator, X is a DNA alkylator, B is aheteroaromatic residue that is attached to the N-terminus of thealkylator subunit (X), Y is a linker, T and T′ are the same or differentand each is a terminal subunit, Z is a polyamide group that binds to theminor groove of DNA, and a is 0 or 1, provided that when V isnaphthalimide, then Z is not lexitropsin, and further provided that whenV is doxorubicin, then Z is not netropsin or distatnycin. In addition,the present invention provides a conjugate of formula:V—(Y)_(a)-Z-(Y′)_(a)—V′  (III),T-X—B —(Y)_(a)-Z-(Y′)_(a)—X″—B′-T′  (IV) orV—(Y)_(a)-Z-(Y′)_(a)—X—B-T  (V),in which V and V′ are the same or different and each is a DNAintercalator, X and X′ are the same or different and each is a DNAalkylator, B and B′ are the same or diffent and each is a heteroaromaticresidue that is attached to the N-terminus of an alkylator subunit (X orX′), Y and Y′ are the same or different and each is a linker, T and T′are the same or different and each is a terminal subunit, Z is apolyamide group that binds to the minor groove of DNA, and a is 0 or 1.The present invention further provides a conjugate of formula:V—(Y)_(a)-Z-X—B-Z′-(Y′)_(a)—(V′)_(b)  (VI),in which V and V′ are the same or different and each is a DNAintercalator, X is a DNA alkylator, B is a heteroaromatic residue thatis attached to the N-terminus of the alkylator subunit (X), Y and Y′ arethe same or different and each is a linker, Z and Z′ are the same ordifferent and each is a polyamide group that binds to the minor grooveof DNA, and a and b are independently 0 or 1.

In addition, the present invention provides a hybrid conjugate offormula:(W)_(a)—(Y)_(b)—[(Z)_(c)-(Y′)_(d)—(X—B)_(e)—(Y″)_(f)-(Z′)_(g)]_(h)—(Y′″)_(i)—(W′)_(j)  (VII),in which W and W′ are the same or different and each is a DNAintercalator (V or V′) or terminal subunit (T or T′), X is a DNAalkylator, B is a heteroaromatic residue that is attached to theN-terminus of the alkylator subunit (X), Y, Y′, Y″ and Y′″ are the sameor different and each is a linker, Z and Z′ are the same or differentand each is a polyamide group that binds to the minor groove of DNA, ais 0-5, b is 0-5, c is 0-5, d is 0-5, e is 1-5, f is 0-5, g is 0-5, h is1-5, i is 0-5, and j is 0-5.

Further provided by the present invention is a composition comprising aconjugate of any of formulae (I)-(VI) and a carrier.

Still further provided by the present invention is a method for treatingcancer in a mammal comprising administering to a mammal in need thereofan effective amount of a conjugate of any of formulae (I)-(VI) or acomposition thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the synthetic scheme of{1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole-8-carboxylicacid} (“CBIr”), a rigid DNA alkylator.

FIG. 2 depicts the convergent solid phase synthesis of a conjugatecomprising{1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole-8-carboxylicacid} (“CBIr”), a rigid DNA alkylator.

FIG. 3 depicts the synthetic scheme of2-{1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indol-8-yl}aceticacid (“CBIf”), a flexible DNA alkylator.

FIG. 4 depicts the convergent solid phase synthesis of a conjugatecomprising2-{1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indol-8-yl}aceticacid (“CBWIf”), a flexible DNA alkylator.

FIG. 5 shows the structures of CBI-AM and CBI-Bf.

FIG. 6A is a graph of % cell growth vs. drug concentration (μM) forCBI-AM in an MTT assay at 120 hr.

FIG. 6B is a graph of % growth vs. drug concentration (μM) for CBI-BF inan MTT assay at 72 hr.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides conjugates that comprise one or morepolyamide minor groove binder(s) linked to one or more DNAintercalator(s) and/or DNA alkylator(s). The conjugates bindspecifically to preselected genes, i.e., coding sequence or controlsequence, such as promoter or enhancer, effectively inhibiting thegenes' functions. Therefore, the conjugates are useful as therapeuticagents in the treatment of cancer, in which a known gene is responsiblefor the survival of the cancer cell.

In one aspect, the invention provides a conjugate of formula (I) or (H):V—(Y)_(a)-Z-T  (I) orT-X—B—(Y)_(a)-Z-T′  (II),in which V is a DNA intercalator, X is a DNA alkylator, B is aheteroaromatic residue that is attached to the N-terminus of thealkylator subunit (X), Y is a linker, T and T′ are the same or differentterminal subunits, Z is a polyamide group that binds to the minor grooveof DNA, and a is 0 or 1, provided that when V is naphthalimide, then Zis not lexitropsin, and further provided that when V is doxorubicin,then Z is not netropsin or distamycin.

In another aspect, the present invention provides a conjugate offormula:V—(Y)_(a)-Z-(Y′)_(a)—V′  (I),T-X—B—(Y)_(a)-Z-(Y′)_(a)—X′—B′-T′  (IV) orV—(Y)_(a)-Z-(Y′)_(a)—X—B-T  (V),in which V and V′ are the same or different and each is a DNAintercalator, X and X′ are the same or different and each is a DNAalkylator, B and B′ are the same or diffent and each is a heteroaromaticresidue that is attached to the N-terminus of an alkylator subunit (X orX′), Y and Y′ are the same or different and each is a linker, T and T′are the same or different and each is a terminal subunit, Z is apolyamide group that binds to the minor groove of DNA, and a is 0 or 1.

In yet another aspect, the present invention provides a conjugate offormula:V—(Y)_(a)-Z-X—B-Z′-(Y′)_(a)—(V′)_(b)  (VI),in which V and V′ are the same or different and each is a DNAintercalator, X is a DNA alkylator, B is a heteroaromatic residue thatis attached to the N-terminus of the alkylator subunit (X), Y and Y′ arethe same or different and each is a linker, Z and Z′ are the same ordifferent and each is a polyamide group that binds to the minor grooveof DNA, and a and b are independently 0 or 1.

In addition, the present invention provides a hybrid conjugate offormula:(W)_(a)—(Y)_(b)—[(Z)_(c)-(Y′)_(d)—(X—B)_(e)—(Y″)_(f)(Z′)_(g)]_(h)—(Y′″)_(i)—(W′)_(j)  (VII),in which W and W′ are the same or different and each is a DNAintercalator (V or V′) or terminal subunit (T or T′), B is aheteroaromatic residue that is attached to the N-terminus of thealkylator subunit (X), Y, Y′, Y″ and Y′″ are the same or different andeach is a linker, Z and Z′ are the same or different and each is apolyamide group that binds to the minor groove of DNA, X is a DNAalkylator, a is 0-5, b is 0-5, c is 0-5, d is 0-5, e is 1-5, f is 0-5, gis 0-5, h is 1-5, i is 0-5, and j is 0-5. Preferably, a, b, c, d, f, g,i, and j are independently 0-4, more preferably 0-3, even morepreferably 0-2, and most preferably 0 or 1. Preferably, e and j areindependently 1-5, more preferably 1-4, even more preferably 1-3, andmost preferably 1 or 2. When h is 2 or higher, compounds of formula(VII) preferably comprise 2 or more polyamide groups (Z, Z′) that arethe same or different and 2 or more alkylators (X, X′) that are the sameor different.

The DNA intercalator (V and/or V′) is any suitable moiety that canintercalate between selected base pairs of DNA, preferably in the minorgroove, and is toxic to the cancer cells to which it binds.Alternatively, the DNA intercalator can form covalent bonds withnucleophilic positions on the DNA bases. Specific examples of DNAintercalators include, but are not limited to, substituted orunsubstituted anthracyclines, such as doxorubicin or daunorubicin;substituted or unsubstituted imidazoacridone; substituted orunsubstituted 3-nitrophthalamide; and substituted or unsubstituted3-aminophthalamide. Any suitable moieties can be used to substitute theaforementioned intercalators, provided that the moieties do notadversely affect the ability of the intercalator to intercalate betweenselected base pairs of DNA or to form covalent bonds with nucleophilicpositions on DNA bases. For example, acceptable substituents include,but are not limited to, hydroxy, C₁₋₁₂ alkoxy, acyloxy, halo or benzyl,acetyl, carboxyl, carboxy-C₁₋₁₂ alkyl, such as carboxymethyl,carboxyethyl, carboxy-C₁₋₁₂ alkylamido, carboxy-C₁₋₁₂ dialkylamido,carboxamido, amino, C₁₋₁₂ alkylamino, C₁₋₁₂ dialkylamino, C₁₋₁₂alkylcarbonyl, C₆₋₃₀ arylamino, C₆₋₃₀ diarylamino, cyano, tolyl, xylyl,mesityl, anisyl, pyrrolidinyl, formyl, dioxane, thio, C₁₋₁₂ alkylthio,C₆₋₃₀ aryl, C₅₋₃₀ heteroaryl, such as pyranyl, pyrrolyl, furanyl,thiophenyl, thiazolyl, pyrazolyl, pyridinyl, or pyrimidinyl, phenoxy,benzyloxy, phenylcarbonyl, benzylcarbonyl, nitrophenyl C₁₋₁₂trialkylsilyl, nitro, sulfonyl, nitrobenzyl, C₁₋₁₂ trialkylammonium,C₁₋₁₂ alkyl, C₃₋₈ cycloalkyl, tetrahydrofuranyl, tetrahydropyranyl,piperidinyl and morpholinyl.

The DNA alkylator (X and/or X′) is any suitable sequence-selectivealkylating agent that is toxic to DNA for which it is selective.Preferably, the DNA alkylator is a rigid alkylator or a flexiblealkylator. In particular, the rigid alkylator is preferably{1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole-8-carboxylicacid} (“CBIr”):

that is optionally further substituted, and the flexible alkylator is2-{1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indol-8-yl}aceticacid (“CBIf”):

that is optionally further substituted, or8-(aminomethyl)-1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole(“CBIa”):

that is optionally further substituted. These compounds are part of aclass of compounds based on a cyclopropanobenzindole (CBI) skeleton thatis known to be extremely cytotoxic (e.g., IC₅₀ in the nano- to picomolarrange). With respect to CBI analogs, preferably the linking moiety orpolyamide groove binder is connected at the C3 and C7 positions, asshown in the following structure:

Conjugates in which the connectivity of the CBI analog is through the C3and C8 positions are generally not preferred because the geometry of theconjugate is not ideal for fitting in the minor groove of DNA. The CBIanalogs described above comprise an amino acid or amino alkyl moiety,thereby allowing them to be part of the polyamide minor groove bindersequence. Therefore, conjugates that comprise such amino acid or aminoalkyl DNA alkylators do not necessarily require an additional linker tolink the alkylator to the polyamide minor groove binder.

It is understood for purposes of this invention that the CBI moiety canbe in an “open” form or a “closed” form as follows:

x=halide, esters such as but not limited to mesylate, tosylate,nitroureas, nitrocarbamates. Their capacity to alkylate DNA is virtuallyequivalent.

The alkylator subunits have been designed so that they can beincorporated into various positions of the DNA sequence-recognizingminor groove binders. In other words, the CBI derivatives function asconstituent units of the minor groove binding structure. This is unlikethe CBI derivatives designed earlier, which can be attached to theC-terminal or N-terminal end of the polyamide chains but not within thechain. The difference between the present inventive CBI derivatives andthe other constituent units of the DNA-recognizing sequences is thatthey react covalently with the DNA in a highly sequence-specific mannerimparted by their particular placement within the sequence. Besides theincreased specificity of binding, the embedded CBI units can decreasethe chances of unspecific alkylation of DNA, which is more likely whenCBI-terminated binders (at either ends) interact with DNA.

The polyamide group that binds to the minor groove of DNA (Z and/or Z′)is any suitable polyamide sequence that recognizes specific DNAsequences through minor groove binding. Preferably, the polyamidesequence comprises one or more substituted or unsubstituted polypyrrolecarboxamide, one or more substituted or unsubstituted polyimidazolecarboxamide, or combinations thereof. More preferably, the polyamideminor groove binder comprises subunits of4-amino-1-methylpyrrole-2-carboxylic acid,4-amino-1-methylimidazole-2-carboxylic acid,4-amino-1-methyl-3-hydroxypyrrole-2-carboxylic acid, γ-amino-butyricacid, α,γ-diamino-butyric acid, glutamic acid, 8-amino-3,6-dioxanioicacid, β-alanine, 4-amino-benzoic acid, 3-amino-benzoic acid,2-aminothiazole-5-carboxylic acid, 4-aminothiophene-2-carboxylic acid,5-aminobenzthiophene-2-carboxylic acid, 5-aminobenzoxazole-2-carboxylicacid, 5-aminobenzimidazole-2-carboxylic acid or combinations thereof. Itis understood that the longer the polyamide sequence, the moreDNA-selective it is considered to be. The polyamide minor groove binderpreferably contains 20 or fewer amide subunits, more preferably 10 orfewer amide subunits, more preferably 8 or fewer amide subunits, morepreferably 6 or fewer amide subunits, and most preferably 5 or feweramide subunits. A preferred conjugate will contain one or more polyamideminor groove binder(s) made up of 4 or 5 amide subunits.

The heteroaromatic residue (B) that is attached to the N-terminus of thealkylator subunit (X or X′) increases the activity of the alkylator,resulting in an agent with much higher cytotoxicity. Preferably, anyunit listed for Z (minor groove binder) is suitable for the B subunit.More preferably, bicyclic or tricyclic molecules, such as indoles,benzofuranes and the like, should be used.

Conjugates of the present invention can comprise one polyamide minorgroove binder (i.e., single-stranded) or more than one polyamide minorgroove binder (e.g., double-stranded). In the case of double-strandedconjugates, the conjugate can optionally form a hairpin structure.Without wishing to be bound to any particular theory, it is believedthat an entire hairpin structure can fit into the minor groove and hasmuch stronger binding than its corresponding single-stranded conjugate.

The linker comprises any moiety that can form a chemical bond betweenthe polyamide minor groove binder (i.e., Z and/or Z′) and theintercalator (V and/or V′) or the DNA alkylator (X and/or X′). Thelinker can optionally bind to the minor groove of DNA but can havelittle to no ability for sequence-specific interaction. The linker canbe of any suitable charge, length and/or rigidity, but preferably thelinker is bifunctional and/or comprises one or more amino groups. Atphysiological pH, the amino group is protonated and can also bind toDNA. The amino group can be primary, secondary or tertiary. Preferably,the amino group comprises a moiety selected from the group consisting ofamino, C₁-C₁₂ alkylamino, C₁-C₁₂ dialkylamino, cycloalkylamino,piperazinyl, piperidinyl, pyrazinyl, purinyl, pyridazinyl, pyrrolidinyl,oxazolyl, isooxazolyl, quinolinyl, isoquinolinyl, byrimidinyl,morpholinyl, thiazolyl, isothiazolyl, quinoxalinyl, quinazolinyl,pyrrolyl, imidazolyl and an amino acid residue. Specific examples ofsuitable linkers include, but are not limited to,N,N′-bis(aminopropyl)piperazine, N,N′-bis(aminopropyl)methylamine,8-amino-3,6-dioxaoctanoic acid, spermidine and β-alanine.

The conjugate can be optionally terminated at either end with a moietythat forms a chemical bond with either the N-terminus or the C-terminusof the conjugate. The terminal subunit (T and/or T′) can comprise anysuitable functional group, but preferably contains at least one aminogroup, amidine, guanidine or a carboxamide moiety. At physiological pH,the amino group is protonated and can also bind to the DNA sequence.Typical terminal subunits include, but are not limited to,N,N-dimethylglycine, guanidino acetic acid, 3-aminopropylamidine,glycinol, N,N-dimethylaminopropylamine, N-formyl, N-acetyl, N-propionyland N-benzoyl.

The conjugate of any of formulae (I)-(VII) binds to DNA, preferably inthe minor groove. The conjugate is sequence-selective and binds to atleast 5 base pairs in DNA, preferably at least 7 base pairs in DNA, morepreferably at least 9 base pairs in DNA, and most preferably at least 10base pairs in DNA. Depending on the specific polyamide minor groovebinder and intercalator and/or alkylator selected for the conjugate, theconjugate can bind to a G-C rich DNA sequence or to an A-T rich DNAsequence. Those skilled in the art will understand how to make suchselections for each element of the conjugate in order to bind to a G-Cor A-T rich DNA sequence. For example, DNA alkylators such as CBIanalogs, as described herein, bind covalently to adenine residuesthrough the minor groove. Therefore, conjugates comprising a CBI analogcan be used to bind selectively to A-T rich DNA sequences.Alternatively, conjugates that comprise a phthalimide or anthracyclineresidue tend to favor G-C rich DNA sequences. The selection of subunitsof the polyamide minor groove binder allows for the preparation ofsequences that can preferentially bind to G-C or A-T rich DNA sequences.For example, N-methylimidazole preferably will bind to guanosine,whereas N-methylpyrrole preferably will bind to cytosine, adenine, andthymidine.

A preferred conjugate of formula (I) is:

in which Y is a linker; in each repeat unit, Q is independently N or CH;R¹ and R² are selected from the group consisting of hydrogen, C₁-C₁₂alkyl, hydroxy, and halo; and n and m are independently 1 to 6. Anotherpreferred conjugate of formula (I) is:

in which R³ is —NO₂ or —NH₂, Y is a linker; in each repeat unit, Q isindependently N or CH; R¹ and R² are selected from the group consistingof hydrogen, C₁-C₁₂ alkyl, hydroxy, and halo; and n and m areindependently 1 to 6. An especially preferred example of a conjugate offormula (I) is:

Another preferred conjugate of formula (I) is:

in which in each repeat unit, Q is independently N or CH; R¹ and R² areselected from the group consisting of hydrogen, C₁-C₁₂ alkyl, hydroxy,and halo; and n and m are independently 1 to 6, provided that when Q ineach repeat unit is CH, R¹ is CH₃ and R² is H, then n is not 1-3. Anespecially preferred conjugate of formula (I) is:

A preferred conjugate of formula (II) is:

in which Y is a linker; in each repeat unit, Q is independently N or CH;B is a heteroaromatic residue as defined above; T is a terminal subunitas defined above; R¹ and R² are selected from the group consisting ofhydrogen, C₁-C₁₂ alkyl, hydroxy, and halo; n and m are independently 1to 6; and 0 is 0 or 1. An especially preferred example of a conjugate offormula (II) is:

Another preferred conjugate of formula (II) is:

in which Y is a linker; in each repeat unit, Q is independently N or CH;B is a heteroaromatic residue as defined above; T is a terminal subunitas defined above; R¹ and R² are selected from the group consisting ofhydrogen, C₁-C₁₂ alkyl, hydroxy, and halo; and n and m are independently1 to 6.

A preferred conjugate of conjugate of formula (III) is:

in which Y and Y′ can be the same or different and each is a linker; ineach repeat unit, Q is independently N or CH; R¹ and R² are selectedfrom the group consisting of hydrogen, C₁-C₁₂ alkyl, hydroxy, and halo;and n is 1 to 6. An especially preferred conjugate of formula (II) is:

A preferred conjugate of formula (IV) is:

in which Y and Y′ can be the same or different and each is a linker; ineach repeat unit, Q is independently N or CH; B and B′ are the same ordifferent and each is a heteroaromatic residue as defined above; T is aterminal subunit as defined above; R¹ and R² are selected from the groupconsisting of hydrogen, C₁-C₁₂ alkyl, hydroxy, and halo; n is 1 to 6;and o and o′ are independently 0 or 1. An especially preferred conjugateof formula (IV) is:

A preferred conjugate of formula (V) is:

in which B is a heteroaromatic residue as defined above; T is a terminalsubunit as defined above; Y and Y′ can be the same or different and eachis a linker, in each repeat unit, Q is independently N or CH; R¹ and R²are selected from the group consisting of hydrogen, C₁-C₁₂ alkyl,hydroxy, and halo; n is 1 to 6; and o is 0 or 1. An especially preferredconjugate of formula (V) is:

A preferred conjugate of formula (VI) is:

in which Y and Y′ can be the same or different and each is a linker; Bis a heteroaromatic residue as defined above; in each repeat unit, Q isindependently N or CH; R¹⁴ are selected from the group consisting ofhydrogen, C₁-C₁₂ alkyl, hydroxy, and halo; n and p independently are 1to 6; and o is 0 or 1. An especially preferred conjugate of formula (VI)is:

Another especially preferred conjugate of formula (VI) is:

A preferred conjugate of formula (VI), (in which a=b=e=hj=l andc=d=f=g=i=0) is:

The imidazoacridine moriety can be substituted in position 8 by a C₁-C₆alkyl or a C₁-C₆alkoxy. Specific examples of such substituents includemethyl, methoxyl, hydroxylmethyl and the like.

Another especially preferred conjugate of formula (VII) (in whicha=e=h=j=l and b=c=d=f=g=i=0) is:

Although these two compounds do not include a polyamide sequence forminor groove binding, the CBIr and CBIf subunits have minor groovebinding/recognition characteristics, so they are encompassed asDNA-intercalator—minor groove binder conjugates.

Another especially preferred conjugate of formula (VII) representing thegroup, of alkylator oligomers (in which e=3, a=h=j=1 and b=c=d=f=g=i=0)is:

Another especially preferred conjugate of formula (VII) (in whicha=b=c=d=e=f=g=i=j=1 and h=2) is:

To increase the selectivity and efficacy of the conjugates of formulae(I)-(VII) double stranded hairpin polyamide group might be used in anyof the conjugates. For example, the following conjugate has a folded,hairpin structure:

With respect to the above conjugates, a C₁-C₁₂ alkyl can be straightchain or branched chain. In addition, the C₁-C₁₂ alkyl can be optionallysubstituted with substituents such as, for example, hydroxy, C₁₋₁₂alkoxy, acyloxy, halo or benzyl, acetyl, carboxyl, carboxy-C₁₋₁₂ alkyl,such as carboxymethyl, carboxyethyl, carboxy-C₁₋₁₂ alkylamido,carboxy-C₁₋₁₂ dialkylamido, carboxamido, amino, C₁₋₁₂ alkylamino, C₁₋₁₂dialkylamino, C₁₋₁₂ alkylcarbonyl, C₆₋₃₀ arylamino, C₆₋₃₀ diarylamino,cyano, tolyl, xylyl, mesityl, anisyl, pyrrolidinyl, formyl, dioxane,thio, C₁₋₁₂ alkylthio, C₆₋₃₀ aryl, C₅₋₃₀ heteroaryl, such as pyranyl,pyrrolyl, furanyl, thiophenyl, thiazolyl, pyrazolyl, pyridinyl, orpyrimidinyl, phenoxy, benzyloxy, phenylcarbonyl, benzylcarbonyl,nitrophenyl C₁₋₁₂ trialkylsilyl, nitro, sulfonyl, nitrobenzyl, C₁₋₁₂trialkylammonium, C₁₋₁₂ alkyl, C₃₋₈ cycloalkyl, tetrahydrofuranyl,tetrahydropyranyl, piperidinyl and morpholinyl. Typical examples of aC₁-C₁₂ alkyl are methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl,s-butyl, t-butyl, pentyl, neo-pentyl, hexyl, octyl, decyl; etc.

The conjugates of formulae (I)-(VI) can be prepared by any suitablemethod. The examples included herein are merely exemplary methods, andthe synthetic routes for the conjugates are in no way limited to thesemethods. Those ordinarily skilled in the art will know that thesynthesis of some moieties, such as some DNA intercalators or DNAalkylators, can be prepared by methods recited in the literature.

Although not required, the conjugates are preferably prepared byheterogeneous methods using solid supports. In general, the minor groovebinder oligomers are synthesized using solid-phase synthesis (see, forexample, Dervan et al., Org. Lett., 3: 1201-1203 (2001)). Depending onthe post-solid-phase steps (e.g., coupling the minor groove binders todoxorubicin), different resins are used, such as, for example,Wang-resin, hydroxymethyl-benzoic acid resin, trityl-resin; etc.

Preferably, all of the monomers (e.g., pyrrole-, imidazole-amino acidsand alkylator subunits) are introduced as protected residues (e.g.,Fmoc-protected). Piperidine or piperidine/DBU typically are used todeprotect the monomers. For coupling, the monomers are preferablypreactivated or activated in situ. In the case of aromatic carboxylicacids, stable benzotriazyl active esters were isolated for the couplingreaction, whereas, in the case of aliphatic carboxylic acids, variousactivating agents (e.g., HBTU, HATU, HOBT/DCC) were used for activation.In addition to benzotriazyl esters, aromatic carboxylic acids can beactivated by oxalyl chloride in the presence of a catalytic amount ofDMF and used as acyl-chlorides for coupling reactions. The alkylatorresidue CBIr, CBIf, or CBIa is preferably modified to form the pro-drugequivalent (seco-form) in three steps: (i) removal of the TBDMS group;(ii) converting the alcohol into a chloro group; and (iii) deprotectionof the t-Butyl ether to release the phenol. The oligomer is generallycleaved from the resin using either acidolytic (e.g., Wang- or tritylresins) or nucleophilic (e.g., HMBA resin) conditions. The intercalatorsubunit preferably is introduced just prior to the cleavage from theresin or after the cleavage in a solution-phase step.

In another aspect, the invention provides compositions, includingpharmaceutical compositions, comprising the conjugate of formula (I),(II), (III), (IV), (V), (VI), or (VII) or combinations thereof, and acarrier, alone or in further combination with other active agents, suchas adjuvants and anti-cancer agents. Preferably, the pharmaceuticalcompositions further comprise a pharmaceutically acceptable carrier.

One ordinarily skilled in the art will appreciate that suitable methodsof administering a conjugate or composition thereof to a mammal, such asa human, are known, and, although more than one route can be used toadminister a particular composition, a particular route can provide amore immediate and more effective reaction than another route. If thecancer is in the form of a tumor, preferably the conjugate orcomposition thereof is administered intratumorally or peritumorally.Pharmaceutically acceptable carriers are also well-known in the art. Thechoice of carrier will be determined, in part, by the particularconjugate or composition and by the particular method used to administerthe composition. Accordingly, there are a wide variety of suitableformulations of the pharmaceutical compositions of the presentinvention.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the conjugate of any offormulae (I)-(VII) dissolved in diluents, such as water or saline, (b)capsules, sachets or tablets, each containing a predetermined amount ofthe active ingredient, as solids or granules, (c) suspensions in anappropriate liquid, and (d) suitable emulsions.

Tablet forms can include one or more of lactose, mannitol, cornstarch,potato starch, microcrystalline cellulose, acacia, gelatin, colloidalsilicon dioxide, croscarmellose sodium, talc, magnesium stearate,stearic acid, and other excipients, colorants, diluents, bufferingagents, moistening agents, preservatives, flavoring agents, andpharmacologically compatible carriers. Lozenge forms can comprise theactive ingredient in a flavor, usually sucrose and acacia or tragacanth,as well as pastilles comprising the active ingredient in an inert base,such as gelatin and glycerin or sucrose and acacia emulsions, gels, andthe like containing, in addition to the active ingredient, such carriersas are known in the art.

The conjugates of the present invention, alone or in combination withother suitable components, can be made into aerosol formulations to beadministered via inhalation. These aerosol formulations can be placedinto pressurized acceptable propellants, such asdichlorodifluoromethane, hydrofluorocarbon (such as HFC 134a and/or227), propane, nitrogen and the like.

Formulations suitable for parenteral administration include aqueous andnon-aqueous solutions, isotonic sterile injection solutions, which cancontain anti-oxidants, buffers, bacteriostats and solutes that renderthe formulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers and preservatives.The formulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example, water, for injections, immediatelyprior to use. Extemporaneous injection solutions and suspensions can beprepared from sterile powders, granules, and tablets of the kindpreviously described.

The dose administered to a mammal, particularly a human, in the contextof the present invention should be sufficient to effect a therapeuticresponse in the mammal over a reasonable time frame. The dose will bedetermined by the strength of the particular composition employed(taking into consideration, at least, the bioactivity of anydecomposition products derived from the conjugates) and the condition ofthe mammal (e.g., human), as well as the body weight of the mammal(e.g., human) to be treated. The size of the dose also will bedetermined by the existence, nature, and extent of any adverse sideeffects that might accompany the administration of a particularcomposition. A suitable dosage for internal administration is 0.01 to100 mg/kg of body weight per day, such as 0.01 to 35 mg/kg of bodyweight per day or 0.05 to 5 mg/kg of body weight per day. A suitableconcentration of the conjugate in pharmaceutical compositions fortopical administration is 0.05 to 15% (by weight), preferably 0.02 to5%, and more preferably 0.1 to 3%.

The conjugates of any of formulae (I)-(VI) or compositions thereof areuseful for treating a mammal, such as a human, for cancer. The methodcomprises administering to the mammal, e.g., human, a cancer-treatmenteffective amount of a conjugate of any of formulae (I)-(VII) or acomposition thereof, whereupon the mammal is treated for cancer. Thetreatment can be prophylactic or therapeutic. By “prophylactic” is meantany degree in inhibition of the onset of cancer, including completeinhibition. By “therapeutic” is meant any degree in inhibition of thegrowth or metastasis of the cancer in the mammal (e.g., human).

The method can be used in combination with other known treatmentmethods, such as radiation, surgery, or the administration of otheractive agents, such as adjuvants or other anti-cancer agents and theirprodrugs. Examples of cyotoxic agents and their prodrugs includegenistein, okadaic acid, 1-β-D-arabinofuranosyl-cytosine,arabinofuranosyl-5-aza-cytosine, cisplatin, carboplatin, actinomycin D,asparaginase, bis-chloro-ethyl-nitroso-urea, bleomycin, chlorambucil,cyclohexyl-chloro-ethyl-nitroso-urea, cytosine arabinoside, daunomycin,etoposide, hydroxyurea, melphalan, mercaptopurine, mitomycin C, nitrogenmustard, procarbazine, teniposide, thioguanine, thiotepa, vincristine,5-fluorouracil, 5-fluorocytosine, adriamycin, cyclophosphamide,methotrexate, vinblastine, doxorubicin, leucovorin, taxol, anti-estrogenagents such as tamoxifen, intracellular antibodies against oncogenes,the flavonol quercetin, Guan-mu-tong extract, retinoids such asfenretinide, nontoxid retinoid analogues such asN-(4-hydroxyphenyl)-retinamide (HPR), and monoterpenes such as limonene,perillyl alcohol and sobrerol.

The method of treating cancer with a conjugate of any of formulae(I)-(VII) or composition thereof can be combined with still othermethods of prophylactic and therapeutic treatment. Such methods includethose that target destruction of cancer cells, e.g., by targeting ofcell-surface markers, receptor ligands, e.g., ligands togastrin-releasing peptide-like receptors, tumor-associated antigens,e.g., the 57 kD cytokeratin or the antigen recognized by the monoclonalantibody GB24, the extracellular matrix glycoprotein tamascin, DNAreplication, the transcription of genes whose protein products arenecessary for the survival of the cancer, e.g., proteins involved insignal transduction, growth factor receptors, nuclear oncoproteins,antagonists of apoptosis, antagonists of tumor suppressors, and proteinsinvolved in DNA repair processes, antisense oncogenes such as c-fos,homeobox genes that are expressed in cancer cells but not normal cells,tumor-infiltrating lymphocytes that express cytokines, RGD-containingpeptides and proteins, which are administered following surgery,lipophilic drug-containing liposomes to which are covalendy conjugatedmonoclonal antibodies for targeting to cancer cells, low fat diet,moderate physical exercise and hormonal modulation. For prostate cancer,anti-testosterone agents can be used as well as an inhibitor of cellularproliferation produced by prostatic stromal cells and C-CAM, anepithelial cell adhesion molecule.

The conjugates of any of formulae (I)-(VII) preferably are useful fortargeting coding regions or control regions of genes, such as promotersor enhancers, and inhibiting transcription. Cancers that are suitable totreatment with conjugates of the present invention include those inwhich specific genes are known to be mutated and/or over-expressed andnecessary for the survival of the cancer cell. Preferably, the molecularpathogenesis of the cancer involves one or more potential moleculartargets: (i) molecules involved in signal transduction (e.g., K-Ras);(ii) growth factor receptors (e.g., Her-2-Neu); (iii) nuclearoncoproteins (e.g., c-Myc); (iv) antagonists of apoptosis (e.g., BCL2);and (v) antagonists of tumor suppressors (e.g., MDM2); (vi) DNA repairprocesses; and (vii) DNA transcription and replication. For instance,typical cancers that are to be treated in conduction with the conjugatesand compositions of the present invention incude breast cancer,metastatic melanoma, follicular thyroid carcinoma, colorectal cancer,pancreatic cancer, leukemias, such as myeloid leukemia, prostate cancer,hepatic cancer, hepatocellular carcinoma, cholangiocarcinoma, cervicaland ovarian cancer, cancers of glial origin and renal cancer.

EXAMPLES

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

The following abbreviations are used herein:

-   BOC or Boc i-Butyloxycarbonyl,-   Bn Benzyl,-   Bt 1H-Benztriazol-1-yl,-   ^(t)Bu t-Butyl,-   DBU 1,8-Diazabicyclo[5.4.0]undec-7-ene,-   DCC 1,3-Dicyclohexylcarbodiimide,-   DCM Dichloromethane,-   DIPEA Diisopropylethylamine,-   DPPA Diphenylphosphoryl azide,-   FMOC or Fmoc 9-Fluorenylmethoxycarbonyl,-   FmocCl 9-Fluorenylmethoxycarbonylchloride,-   Fmoc-Im-OBt (1H-Benzotriazole-1-yl)    4-(9-fluorenylmethoxycarbonyl)amino-1-methylimidazole-2-carboxylate,-   Fmoc-Py-OBt (1H-Benzotriazole-1-yl)    4-(9-fluorenylmethoxycarbonyl)amino-1-methylpyrrole-2-carboxylate,-   HATU 2-(1H-7-Azabenzotriazole-1-yl)-1,1,3,3-tetramethyluronium    hexafluorophosphate,-   HBTU 2(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium    hexafluorophosphate,-   HFIP Hexafluoroisopropanol,-   HMBA Hydroxymethylbenzoicacid,-   HOAt N-Hydroxy-7-azabenzotriazole,-   HOBt N-Hydroxybenzotriazole,-   Im Imidazole-   NIS N-Iodosuccinimide,-   NMP N-Methylpyrrolidone,-   Py pyrrole,-   OBt 1-oxy-benztriazol,-   TBDMS t-Butyldimethylsilyl,-   TBDMSCl t-Butyldimethylsilylchloride,-   TBTH Tributyltinhydride,-   t-BuOH t-Butanol,-   TEMPO 2,2,6,6-tetramethyl-1-piperidinyloxy,-   TFA Trifluoroacetic acid,-   TsOH p-Toluenesulfonic acid,-   WSC 1-Ethyl-3(3′-dimethylaminopropyl)carbodiimide. HCl    (water-soluble carbodiimide), and-   solvent (m×n) washing the resin with a solvent for ‘m’ times during    ‘n’ min.

Example 1

This examples describes the synthesis of(1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole-8-carboxylicacid) (“CBlr”), a rigid DNA alkylator (FIG. 1).

The synthesis procedure for compound 1{5-(Benzyloxy)-3-(tert-butyloxycarbonyl)-8-cyano-1-[(2′,2′,6′,6′-tetramethylpiperidino)oxy]methyl-1,2-dihydro-3H-benz[e]indole}followed that described by Boger (J. Org. Chem. 61: 4894-4912 (1996)).The subsequent synthetic steps were developed to achieve the synthesisof 8 and 9. These steps are described below.

Benzyl5-(benzyloxy)-3-tert-butyloxycarbonyl)-1-[(2′,2′,6′,6′-tetramethylpiperidino)oxy]methyl-1,2-dihydro-3H-benz[e]indole-8-carboxylate(2)

A solution of 1 (1.72 g, 2.33 mmol, 1.0 equiv) in 2-methoxyethanol (6mL) in a round-bottom flask was treated with CsOH (monohydrate, 1.96 g,11.65 mmol, 5.0 equiv) and brought to reflux under a N₂ stream. After 30min, the temperature was decreased to 105° C. and the reaction mixturewas stirred for 14 hr. At this point, no more ammonia was detected byindicator paper in the effluxed N₂. The reaction mixture was then cooledto 25° C., and the volatiles were removed in vacuo. The dark brown solidresidue was dried overnight in high vacuum. Solid CO₂ was added to thesolution of the crude reaction product in acetonitrile (10 mL) at 25° C.The mixture was cooled to 0° C., treated with benzyl bromide (0.554 mL,4.66 mmol, 2.0 equiv) and stirred for 1 hr. The reaction mixture thenwas warmed to 50° C. and was stirred for 30 min and cooled to 25° C. 10%aqueous KHSO₄ and EtOAc were added, and the aqueous layer was extractedwith EtOAc, and the combined organic extract was washed with water andsaturated aqueous NaCl, dried (MgSO₄), and concentrated in vacuo. Flashchromatography (SiO₂, 2.5×12 cm, 0-25% EtOAc-hexane gradient) provided 2(1.29 g, 82%) as an off-white solid.

ES-MS m/z 679.3 (M+H⁺, C₄₂H₅₁N₂O₆ requires m/z 679.3747).

Benzyl5(benzyloxy)-3-(9H-fluoren-9-ylmethoxycarbonyl)-1-[(2′,2′,6′,6′-tetramethylpiperidino)oxy]methyl-1,2-dihydro-3H-benz[e]indole-8-carboxylate(3)

A solution of 2 (1.15 g, 1.69 mM) in DCM (4 mL) was cooled to 0° C.Aqueous TFA (90%; 15 mL) was cooled to 0° C. The TFA solution was addedto the DCM solution drop-wise with stirring for 30 min. The solution wasthen slowly warmed up to 25° C. and was stirred for 20 min. Thevolatiles were removed in vacuo. The trifluoroacetate salt was dried for3 hr in high vacuum and dissolved in anhydrous DMF (8 mL) under argon.The solution was cooled to 0° C. and treated with DIPEA (1.18 mL, 6.76mM, 4.0 equiv), and a solution of Fmoc-Cl (0.456 g, 1.77 mM, 1.05equiv), in anhydrous DMF was added during the period of 10 min. Thereaction mixture was allowed to warm up to 25° C. The DMF was evaporatedin vacuo, and the solid residue was dissolved in EtOAc. The solution wasextracted with H₂O, and the combined aqueous extract was re-extractedwith EtOAc. The combined organic extract was washed with water andsaturated aqueous NaCl, dried (MgSO₄), and concentrated in vacuo. Flashchromatography (SiO₂, 2.5×12 cm, 0-25% EtOAc-hexane gradient) provided 3(1.23 g, 91%) as an off-white solid.

ES-MS m/z 801.4 (M+H⁺, C₅₂H₅₃N₂O₆ requires m/z 801.3903).

Benzyl5-(benzyloxy)-3-(9H-fluoren-9-ylmethoxycarbonyl)-1-(hydroxymethyl)-1,2-dihydro-3H-benz[e]indole-8-carboxylate(4)

A solution of 3 (1.05 g, 1.31 mmol, 1.0 equiv) in THF-HOAc (3:1, 30 mL)under flowing N₂ was treated with activated Zn powder (3.43 g, 40 equiv)and warmed to 75° C. It was then treated with H₂O (8 mL), and themixture was stirred for 6 hr. The Zn powder was removed by filtrationand washed with THF (10 mL) 3 times. The combined filtrate wasevaporated in vacuo and dissolved in EtOAc. The EtOAc solution waswashed with 10% aqueous KHSO₄, water and saturated aqueous NaCl, dried(MgSO₄), and concentrated in vacuo. Flash chromatography (SiO₂, 2.5×12cm, 10-40% EtOAc-hexane gradient) provided 4 (746 mg, 86%) as anoff-white solid.

ES-MS m/z 662.2 (M+H⁺, C₄₃H₃₆N₁O₆ requires m/z 662.2542).

Benzyl5-(benzyloxy)-3-(9H-fluoren-9-ylmethoxycarbonyl)-(tert-butyl-dimethyl-silanyloxymethyl)-1,2-dihydro-3H-benz[e]indole-8-carboxylate(5)

A solution of 5 (720 mg, 1.09 mM, 1.0 equiv) in anhydrous DMF (3 mL)under argon was cooled to 0° C. treated with DIPEA (0.492 mL, 2.83 mM,2.6 equiv) and with a solution of TBDMS-Cl (214 mg, 1.417 mM, 1.3 equiv)in anhydrous DMF (1 mL). The reaction mixture was then slowly warmed upto 25° C. and was stirred for 20 min. The volatiles were removed invacuo and the solid residue was dissolved in EtOAc. The solution wasextracted with H₂O twice, and the combined aqueous extract wasre-extracted with EtOAc. The combined organic extract was washed withwater and saturated aqueous NaCl, dried (MgSO₄), and concentrated invacuo. Flash chromatography (SiO₂, 2.5×12 cm, 0-20% EtOAc-hexanegradient) provided 5 (795 mg, 94%) as a light yellow solid.

ES-MS m/z 776.3 (M+H⁺, C₄₉H₄₉NO₆Si requires m/z 776.3407).

5-(tert-Butyl-dimethyl-silanyloxy)-3-(9H-fluoren-9-ylmethoxycarbonyl)-1-(tert-butyl-dimethyl-silanyloxymethyl)-1,2-dihydro-3H-benz[e]indole-8-carboxylicAcid (7)

A solution of 5 (750 mg, 0.97 mM, 1 equiv) in anhydrous EtOAc/EtOH (1:1v/v, 10 mL) was treated with a slurry of 10% Pd/C (250 mg) in anhydrousEtOH (5 mL). The resulting slurry was degassed with a stream of N₂ for 1min. The reaction mixture was placed under an atmosphere of H₂ andstirred for 6 hr. THF (50 mL) was added to the reaction mixture andfiltered through Celite. The Celite was washed with THF 5 times (15 mLeach). The volatiles of the combined filtrates were removed in vacuo.The resulting slurry was dried under high vacuum over night. Thedebenzylated intermediate 6 was dissolved in anhydrous DMF (5 mL),stirred, cooled to 0° C., and treated with DIPEA (0.843 mL, 4.85 mM, 5equiv) and a solution of TBDMS-Cl (365 mg, 2.43 mM, 2.5 equiv) inanhydrous DMF (1 mL). The reaction mixture was then slowly warmed to 25°C. and stirred for 40 min. The solvents were removed in vacuo, and thesolid residue was dissolved in 30 mL of THF-AcOH (3:1) and stirred atroom temperature. Water (10 mL) was added to the reaction mixture, andit was stirred for 1 hr. The volatiles were removed in vacuo and thesolid residue was dissolved in EtOAc. The solution was extracted withH₂O twice, and the combined aqueous extract was re-extracted with EtOAc.The combined organic extract was washed with water and saturated aqueousNaCl, dried (MgSO₄), and concentrated in vacuo. Flash chromatography(SiO₂, 2.5×12 cm, 0-20% EtOAc-hexane gradient) provided 6(599 mg, 87%)as a light yellow solid.

ES-MS m/z 710.3 (M+H⁺, C₄₁H₅₁NO₆Si₂ requires m/z 710.3333).

Activation of 7

Method A: 71 mg of 7 (0.1 mM, 1.0 equiv) were dissolved in anhydrousDMF. The solution was treated with 14 mg of HOBt (0.105 mM, 1.05 equiv)and 22 mg of DCC (0.105 mM, 1.05 equiv), and stirred at room temperaturefor 20 min, which resulted in the formation of 8.

Method B: 71 mg of 6 (0.1 mM, 1.0 equiv) were dissolved in anhydrousDMF. The solution was treated with oxalyl chloride (22 μL, 0.25 mM, 2.5eq) and 1 μL of DMF. The reaction mixture was stirred for 12 hr, whichresulted in the formation of 9.

Example 2

This examples describes the convergent solid phase synthesis of aconjugate comprising{1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole-8-carboxylicacid} (“CBfr”), a rigid DNA alkylator (FIG. 2).

Glycinol 2-chlorotrityl resin (Novabiochem, San Diego, Calif.) was usedto synthesize the following polyamide sequence:Propanoyl-Py-Py-CBIr-Im-Im-glycinol. After swelling for 1 hr in NMP, theresin was drained, Fmoc-Im-OBt (3 eq) and DIPEA (5 eq) were added andthe mixture was agitated for 2 hr at 40° C. The resin was washed withNMP 4 times. The Fmoc group was removed by two subsequent treatments of40% (v/v) piperidine in NMP (15 min each) then the resin was extensivelywashed with NMP (6×2 min). The above-described‘coupling-washing-deprotection-washing’ cycle was repeated 4 times tocouple the following residues: Fmoc-Im-OBt, Fmoc-CBIr-OBt, Fmoc-Py-OBt,Fmoc-Py-OBt. To terminate the N-terminal amino group, propionicanhydride (2 eq) and DIPEA (4 eq) were added and the mixture wasagitated for 30 min. After washing with NMP (3×3 min) and DCM (3×3 min),the TMDMS groups were removed by TBAF (0.1 M solution in THF, 2×10 mintreatment) and the alcohol was chlorinated using PPh3 (2 eq) and CCl4 (2eq) for 4 hr. The polyamide was cleaved by two subsequent treatments of30% (v/v) HFIP in DCM (30 min each). The filtered cleavage mixtures werecombined and both HFIP and DCM were removed under reduced pressure. Theproduct was analyzed by analytical HPLC and ES-MS. and purified bypreparative HPLC.

Example 3

This examples describes the synthetic scheme of2-{1-chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indol-8-yl}aceticacid (“CBIf”), a flexible DNA alkylator (FIG. 3).

The synthesis until compound 10 follows the same procedure described byBoger (J. Org. Chem., 61: 48944912 (1996)). Mild alkaline hydrolysis of10 using LiOH provided the acid 11. Curtius rearrangement using DPPA int-BuOH formed the protected amine 12, in which the Br was exchanged toLi. Compound 21 was reacted with t-butyl bromoacetate to produce 13.During this conversion, the proton of the urethane was masked as itspotassium salt using KH. Iodination of 14 by NIS in the presence of acatalytic amount of TsOH followed by allylation with allyl bromide andNaH resulted in 15. Radical cyclization of 16 was carried out using TBTHand TEMPO and resulted in 17. In order to synthesize solid-phasecompatible, amino-acid-like compound 21 or 22, all the protecting groupswere removed and three different protecting groups were introduced inthe following order: (i) removal of the BOC and t-Bu (R¹ in FIG. 3)using HCl in dioxane; (ii) Fmoc protection of the secondary amine; (iii)freeing the alcoholic OH group by Zn/AcOH treatment; (iv) reprotectionof the hydroxyl group as the TBDMS ether, (v) hydrogenolysis of thebenzyl group; and (vi) protection of the resulting phenol as the t-Buether provided 20. Before solid-phase application, 20 was activated tothe benzotriazyl active ester (21) or to the acyl chloride (22).

Example 4

This examples describes the convergent solid phase synthesis of aconjugate comprising2-{1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indol-8-yl}aceticacid (“CBIf”), a flexible DNA alkylator (FIG. 4).

Glycinol 2-chlorotrityl resin Novabiochem, San Diego, Calif.) was usedto synthesize the following polyamide sequence:Propanoyl-Py-Py-CBIf-Im-Im-glycinol. After swelling for 1 hr in NMP, theresin was drained, Fmoc-Im-OBt (3 eq) and DIPEA (5 eq) were added andthe mixture was agitated for 2 hr at 40° C. The resin was washed withNMP 4 times. The Fmoc group was removed by two subsequent treatments of40% (v/v) piperidine in NMP (15 min each) then the resin was extensivelywashed with NMP (6×2 min). The above-described‘coupling-washing-deprotection-washing’ cycle was repeated 4 times tocouple the following residues: Fmoc-Im-OBt, Fmoc-CBIf-OBt, Fmoc-Py-OBt,Fmoc-Py-OBt. To terminate the N-terminal amino group, propionicanhydride (2 eq) and DIPEA (4 eq) were added, and the mixture wasagitated for 30 min. After washing with NMP (3×3 min) and DCM (3×3 min),the TMDMS groups were removed by TBAF (0.1 M solution in THF, 2×10 mintreatment) and the alcohol was chlorinated using PPh3 (2 eq) and CCl4 (2eq) for 4 hr. The polyamide was cleaved by two subsequent treatments of30% (v/v) HFIP in DCM (30 min each). The filtered cleavage mixtures werecombined, and both HFIP and DCM were removed under reduced pressure. Theproduct was analyzed by analytical HPLC and ES-MS and purified bypreparative HPLC.

Example 5

This example demonstrates the cytotoxic effect of the present inventivecompounds against cancerous cells.

In order to determine the cytotoxicity of the compounds, “Cell Titer 96”non-radioactive cell proliferation assay (Mossman, J. Immunol. Meth.65:55 (1983)) was used. Preliminary cytotoxicity data for CBI-amideagainst human colon cancer cell line show that it is 3 orders ofmagnitude (1000-fold) less active than the CBI-Bf molecule in HCT116human colon cancer cell line. The two cell lines have a very similarresponse to these types of drugs. The much higher activity of CBI-Bfillustrates the necessity of extension of the CBI residue by thebicyclic benzofuran. Other bicyclic or tricyclic residues that satisfycertain geometric requirements are also effective promoters of activityof CBI. This result indicates that a CBI-Bf or a similarly activated CBIcan be used as an embedded alkylator in a DNA-sequence selective minorgroove binder.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. Recitation of ranges of values herein are merely intended toserve as a shorthand method of referring individually to each separatevalue falling within the range, unless otherwise indicated herein, andeach separate value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein, isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention unless otherwise claimed. Nolanguage in the specification should be construed as indicating anynon-claimed element as essential to the practice of the invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention. Ofcourse, variations of those preferred embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventors expect skilled artisans to employ suchvariations as appropriate, and the inventors intend for the invention tobe practiced otherwise than as specifically described herein.Accordingly, this invention includes all modifications and equivalentsof the subject matter recited in the claims appended hereto as permittedby applicable law. Moreover, any combination of the above-describedelements in all possible variations thereof is encompassed by theinvention unless otherwise indicated herein or otherwise clearlycontradicted by context.

1. A conjugate of formula:

wherein V and V′ are the same or different and each is doxorubicin,daunorubicin, imidazoacridone, 3-nitrophthalamide or 3-aminophthalamide,B is a heteroaromatic residue comprising a subunit of4-amino-1-methylpyrrole-2-carboxylic acid,4-amino-1-methylimidazole-2-carboxylic acid,4-amino-1methyl-3-hydroxypyrrole-2-carboxylic acid, γ-amino-butyricacid, α,γ-diamino-butyric acid, glutamic acid 8-amino-3,6-dioxanioicacid, β-alanine, 4-amino-benzoic acid, 3-amino-benzoic acid,2-aminothiazole-5-carboxylic acid, 4-aminothiophene-2-carboxylic acid,5-aminobenzthiophene-2-carboxylic acid, 5-aminobenzoxazole-2-carboxylicacid, or 5-aminobenzimidazole-2-carboxylic acid, Y and Y′ are the sameor different and each is a bifunctional linker comprising a groupselected from the group consisting of amino, C₁-C₁₂ alkylamino, C₁-C₁₂dialkylamino, cycloalkylamino, piperazinyl, piperidinyl, pyrazinyl,purinyl, pyridazinyl, pyrrolidinyl, oxazolyl, isooxazolyl, quinolinyl,isoquinolinyl, byrimidinyl, morpholinyl, thiazolyl, isothiazolyl,quinoxalinyl, quinazolinyl, pyrrolyl, imidazolyl and an amino acidresidue, in each repeat unit designated by n or p, Q is independently Nor CR², R¹ and R² are the same or different and each is selected fromthe group consisting of hydrogen, C₁-C₁₂ alkyl, hydroxy, and halogen, nand p are independently 1 to 6, o is 0 or 1, and a and b areindependently 0 to
 1. 2. The conjugate of claim 1, wherein the linker Yor Y′ comprises N,N′ -bis(aminopropyl)piperazine,N,N′-bis(aminopropyl)methylamine, 8-amino-3,6-dioxaoctanoic acid,spermidine or ⊖-alanine.
 3. The conjugate of claim 1, wherein Q is N. 4.The conjugate of claim 1, wherein R¹ is C₁-C₁₂ alkyl.
 5. The conjugateof claim 4, wherein R¹ is methyl.
 6. The conjugate of claim 1, wherein nis 4 or
 5. 7. The conjugate of claim 6, wherein n is
 4. 8. The conjugateof claim 1, wherein o is
 0. 9. The conjugate of claim 1, wherein V andV′ are the same or different and each is doxorubicin or daunonibicin.10. A conjugate of formula:

wherein V and V′ are the same or different and each is a substituteddoxorubicin, a substituted daunorubicin. imidazoacridone. a substituted3-nitrophthalamide or a substituted 3-aminophthalamide. wherein thesubstituent is selected from the group consisting of C₁₋₁₂ alkyl, C₃₋₈cycloalkyl, C₆₋₃₀ aryl, C₅₋₃₀ heteroaryl, tolyl, xylyl, mesityl, anisyl,hydroxy, C₁₋₁₂ alkoxy, phenoxy, benzyloxy, acyloxy, halogen, cyano,benzyl, formyl, phenylcarbonyl, benzylcarbonyl, acetyl, carboxyl,carboxy-C₁₋₁₂ alkyl, carboxy-C₁₋₁₂ alkylamido, carboxy-C₁₋₁₂dialkylamido, carboxamido, amino, C₁₋₁₂ alkylamino, C₁₋₁₂ dialkylamino,C₁₋₁₂ alkylcarbonyl, C₆₋₃₀ arylamino, C₆₋₃₀ diarylamino, mercapto, C₁₋₁₂alkylthio, nitro, nitrophenyl, nitrobenzyl, C₁₋₁₂ trialkylsilyl,sulfonyl, C₁₋₁₂ trialkylammonium, pyrrolidinyl, dioxanyl,tetrahydrofuranyl, tetrahydropyranyl, piperidinyl, and morpholinyl, B isa heteroaromatic residue comprising a subunit of4-amino-1-methylpyrrole-2-carboxylic acid,4-amino-1-methylimidazole-2-carboxylic acid,4-amino-1methyl-3-hydroxypyrrole-2-carboxylic acid, γ-amino-butyricacid, α,γ-diamino-butyric acid, glutamic acid, 8-amino-3,6-dioxanioicacid, β-alanine, 4-amino-benzoic acid, 3-amino-benzoic acid,2-aminothiazole-5-carboxylic acid, 4-aminothiophene-2-carboxylic acid,5- aminobenzthiophene-2-carboxylic acid, 5-aminobenzoxazole-2-carboxylicacid, or 5-aminobenzimidazole-2-carboxylic acid, Y and Y′ are the sameor different and each is a bifunctional linker comprising a groupselected from the group consisting of amino, C₁-C₁₂ alkylamino, C₁-C₁₂dialkylamino, cycloalkylamino, piperazinyl, piperidinyl, pyrazinyl,purinyl, pyridazinyl, pyrrolidinyl, oxazolyl, isooxazolyl, quinolinyl,isoquinolinyl, byrimidinyl, morpholinyl, thiazolyl, isothiazolyl,quinoxalinyl, quinazolinyl, pyrrolyl, imidazolyl and an amino acidresidue, in each repeat unit designated by n or p, Q is independently Nor CR², R¹ and R² are the same or different and each is selected fromthe group consisting of hydrogen, C₁-C₁₂ alkyl, hydroxy, and halogen, nand p are independently 1 to 6, o is 0 or 1, and a and b areindependently 0 to
 1. 11. A conjugate of formula:

wherein V and V′ are the same or different and each is doxorubicin,daunorubicin, or imidazoacridone, B is a heteroaromatic residuecomprising a subunit of 2-carbonyl-1H-indole or 2-carbonyl-benzofuran, Yand Y′ are the same or different and each is a bifunctional linkercomprising a group selected from the group consisting of amino, C₁-C₁₂alkylamino, C₁-C₁₂ dialkylamino, cycloalkylamino, piperazinyl,piperidinyl, pyrazinyl, purinyl, pyridazinyl, pyrrolidinyl, oxazolyl,isooxazolyl, quinolinyl, isoquinolinyl, byrimidinyl, morpholinyl,thiazolyl, isothiazolyl, quinoxalinyl, quinazolinyl, pyrrolyl,imidazolyl and an amino acid residue, in each repeat unit designated byn or p, Q is independently N or CR², R¹ and R² are the same or differentand each is selected from the group consisting of hydrogen, C₁-C₁₂alkyl, hydroxy, and halogen, n and p are independently 1 to 6, o is 0 or1, and a and b are independently 0 to
 1. 12. The conjugate of claim 11,which is


13. The conjugate of claim 12, which is


14. The conjugate of claim 11, wherein B is


15. The conjugate of claim 11, wherein B is


16. A conjugate of formula:

wherein V and V′ are the same or different and each is a substituteddoxorubicin, a substituted daunorubicin, or a substitutedimidazoacridone, wherein the substituent is selected from the groupconsisting of C₁₋₁₂ alkyl, C₃₋₈ cycloalkyl, C₆₋₃₀ aryl, C₅₋₃₀heteroaryl, tolyl, xylyl, mesityl, anisyl, hydroxy, C₁₋₁₂ alkoxy,phenoxy, benzyloxy, acyloxy, halogen, cyano, benzyl, formyl,phenylcarbonyl, benzylcarbonyl, acetyl, carboxyl, carboxy-C₁₋₁₂ alkyl,carboxy-C₁₋₁₂ alkylamido, carboxy-C₁₋₁₂ dialkylamido, carboxamido,amino, C₁₋₁₂ alkylamino, C₁₋₁₂ dialkylamino, C₁₋₁₂ alkylcarbonyl, C₆₋₃₀arylamino, C₆₋₃₀ diarylamino, mercapto, C₁₋₁₂ alkylthio, nitro,nitrophenyl, nitrobenzyl, C₁₋₁₂ trialkylsilyl, sulfonyl, C₁₋₁₂trialkylammonium, pyrrolidinyl, dioxanyl, tetrahydrofluranyl,tetrahydropyranyl, piperidinyl, and morpholinyl, B is a heteroaromaticresidue comprising subunits of 2-carbonyl- 1H-indole or2-carbonyl-benzofuran, Y and Y′ are the same or different and each is abifunctional linker comprising a group selected from the groupconsisting of amino, C₁-C₁₂ alkylamino, C₁-C₁₂ dialkylamino,cycloalkylamino, piperazinyl, piperidinyl, pyrazinyl, purinyl,pyridazinyl, pyrrolidinyl, oxazolyl, isooxazolyl, quinolinyl,isoquinolinyl, byrimidinyl, morpholinyl, thiazolyl, isothiazolyl,quinoxalinyl, quinazolinyl, pyrrolyl, imidazolyl and an amino acidresidue, in each repeat unit designated by nor p, Q is independently Nor CR², R¹ and R² are the same or different and each is selected fromthe group consisting of hydrogen, C₁-C₁₂ alkyl, hydroxy, and halogen, nand p are independently 1 to 6, o is 0 or 1, and a and b areindependently 0 to
 1. 17. A pharmaceutical composition comprising theconjugate of claim 1, and a pharmaceutically acceptable carrier.
 18. Apharmaceutical composition comprising the conjugate of claim 2 and apharmaceutically acceptable carrier.
 19. A pharmaceutical compositioncomprising the conjugate of claim 3 and a pharmaceutically acceptablecarrier.
 20. A pharmaceutical composition comprising the conjugate ofclaim 4 and a pharmaceutically acceptable carrier.
 21. A pharmaceuticalcomposition comprising the conjugate of claim 5 and a pharmaceuticallyacceptable carrier.
 22. A pharmaceutical composition comprising theconjugate of claim 6 and a pharmaceutically acceptable carrier.
 23. Apharmaceutical composition comprising the conjugate of claim 7 and apharmaceutically acceptable carrier.
 24. A pharmaceutical compositioncomprising the conjugate of claim 8 and a pharmaceutically acceptablecarrier.
 25. A pharmaceutical composition comprising the conjugate ofclaim 10 and a pharmaceutically acceptable carrier.
 26. A pharmaceuticalcomposition comprising the conjugate of claim 10 and a pharmaceuticallyacceptable carrier.
 27. A pharmaceutical composition comprising theconjugate of claim 11 and a pharmaceutically acceptable carrier.
 28. Apharmaceutical composition comprising the conjugate of claim 16 and apharmaceutically acceptable carrier.
 29. A method for treating cancer ina mammal comprising administering to a mammal in need thereof acancer-treatment effective amount of the conjugate of claim 10,whereupon the mammal is treated for cancer.
 30. A method for treatingcancer in a mammal comprising administering to a mammal in need thereofa cancer-treatment effective amount of the conjugate of claim 1,whereupon the mammal is treated for cancer.
 31. A method for treatingcancer in a mammal comprising administering to a mammal in need thereofa cancer-treatment effective amount of the conjugate of claim 11,whereupon the mammal is treated for cancer.
 32. A method for treatingcancer in a mammal comprising administering to a mammal in need thereofa cancer-treatment effective amount of the conjugate of claim 16,whereupon the mammal is treated for cancer.